Perfluorooctanoic acid (PFOA) has been implicated in various toxicities including neurotoxicity, genotoxicity, nephrotoxicity, epigenetic toxicity, immunotoxicity, reproductive toxicity, and hepatotoxicity. However, information on the accumulation of PFOA in the intestine and its toxic effects on intestinal epigenetics and tight junction (TJ) genes is sparse. CD1 mice were dosed with PFOA (1, 5, 10, or 20 mg/kg/day) for 10 days, and its accumulation and induced alterations in the expression of epigenetic and tight junction genes in the small intestine and colon were evaluated using LC-MS and qPCR techniques. PFOA reduced the expression levels of DNA methyltransferases () primarily in the small intestine whereas, in the colon, a decrease was observed only at high concentrations. Moreover, ten-eleven translocation genes ( and ) expression was dysregulated in the small intestine, whereas in the colon remained unaffected. The tight junction genes Claudins (), Occludin (), and Tight Junction Protein () were also heavily altered in the small intestine. TJs responded differently across the gut, in proportion to PFOA dosing. Our study reveals that PFOA triggers DNA methylation changes and alters the expression of genes essential for maintaining the physical barrier of intestine, with more profound effects in the small intestine compared to the colon.